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Results for “RNA interference”

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PubMed2006

Induction of accommodation model by combined RNA interference targeting 1,3-galactosyltransferase gene and low-dose GS-IB4 lectin in vitro.

OBJECTIVE: This study sought to mimic the interaction of xenograft endothelial cells and human serum in vitro after successfully silencing the expression of porcine alpha1,3-galactosyltransferase (alpha1,3GT) gene by RNA interference (RNAi), and to investigate the possibility of inducing accommodation in vitro by stimulation of alpha-Gal-specific binding lectin, Griffonia simplicifolia isolectin B4 (GS-IB4) and RNAi. MATERIALS AND METHODS: Various alpha-Gal expression patterns on a pig endothelial cell immortalized line (PED) was achieved by serial doses of small interfering RNA (siRNA) targeting porcinc alpha1,3GT gene. alpha1,3GT-siRNA transfected PEDs were exposed to increasing doses of GS-IB4 lectin (0.5, 2, and 8 microg/mL) for 4 hours before incubation with normal human serum (NHS). Accommodation phenomenon of PEDs in NHS was observed by 51Cr release and antibody/complement binding assays. RESULTS: With combined RNAi and low-dose GS-IB4 stimulation, PEDs remarkably inhibited complement-mediated cytotoxicity, which showed a better protective effect than using RNAi alone. At a concentration of 2 mug/mL, GS-IB4 exhibited the maximum protective effect. The expression of E-selectin on alpha1,3GT-siRNA transfected PEDs did not differ from that on parental PEDs with heat-inactivated NHS (HINHS) stimulation. Combined with GS-IB4 stimulation, however, it inhibited expression of E-selectin, which was GS-IB4 dose dependent, resulting in mean fluorescence intensity values of 98.5, 42.0, and 36.3 at 0.5, 2, and 8 microg/mL. The mRNA expression of the protective gene HO-1 was significantly up-regulated after treatment with RNAi and low-dose of GS-IB4. CONCLUSIONS: Combined RNAi and low-dose GS-IB4 induced pig endothelial cell accommodation in vitro. The level of alpha-Gal expression played an important role in the induction of accommodation.

Animals↗

PubMed2002

Stable suppression of tumorigenicity by virus-mediated RNA interference.

Most human tumors harbor multiple genetic alterations, including dominant mutant oncogenes. It is often not clear which of these oncogenes are continuously required and which, when inactivated, may inhibit tumorigenesis. Recently, we developed a vector that mediates suppression of gene expression through RNA interference. Here, we use a retroviral version of this vector to specifically and stably inhibit expression of only the oncogenic K-RAS(V12) allele in human tumor cells. Loss of expression of K-RAS(V12) leads to loss of anchorage-independent growth and tumorigenicity. These results indicate that viral delivery of small interfering RNAs can be used for tumor-specific gene therapy to reverse the oncogenic phenotype of cancer cells.

Animals↗

PubMed2003

RNA interference is required for normal centromere function in fission yeast.

In plants, animals and fungi, active centromeres are associated with arrays of repetitive DNA sequences. The outer repeats at fission yeast (Schizosaccharomyces pombe) centromeres are heterochromatic and are required for the assembly of an active centromere. Components of the RNA interference (RNAi) machinery process transcripts derived from these repeats and mediate the formation of silent chromatin. A subfragment of the repeat (dg) is known to induce silencing of marker genes at euchromatic sites and is required for centromere formation. We show that the RNAi components, Argonaute (Ago1), Dicer (Dcr1) and RNA-dependent RNA polymerase (Rdp1), are required to maintain silencing, lysine 9 methylation of histone H3 and association of Swi6 via this dg ectopic silencer. Deletion of Ago1, Dcr1 or Rdp1 disrupts chromosome segregation leading to a high incidence of lagging chromosomes on late anaphase spindles and sensitivity to a microtubule poison. Analysis of dg transcription indicates that csp mutants, previously shown to abrogate centromere silencing and chromosome segregation, are also defective in the regulation of non-coding centromeric RNAs. In addition, histone H3 lysine 9 methylation at, and recruitment of Swi6 and cohesin to, centromeric repeats is disrupted in these mutants. Thus the formation of silent chromatin on dg repeats and the development of a fully functional centromere is dependent on RNAi.

Centromere↗

PubMed2005

In vitro and in vivo suppression of GJB2 expression by RNA interference.

Mutations in GJB2 (gap junction protein, beta-2) are the major cause of autosomal recessive non-syndromic hearing loss. A few allele variants of this gene also cause autosomal dominant non-syndromic hearing loss as a dominant-negative consequence of expression of the mutant protein. Allele-specific gene suppression by RNA interference (RNAi) is a potentially attractive strategy to prevent hearing loss caused by this mechanism. In this proof-of-principle study, we identified a potent GJB2-targeting short interfering RNA (siRNA) to post-transcriptionally silence the expression of the R75W allele variant of GJB2 in cultured mammalian cells. In a mouse model, this siRNA duplex selectively suppressed GJB2(R75W) expression by >70% of control levels, thereby preventing hearing loss. The level of endogenous murine Gjb2 expression was not affected. Our data show that RNAi can be used with specificity and efficiency in vivo to protect against hearing loss caused as a dominant-negative consequence of mutant gene expression.

Animals↗

PubMed2005

Probing the role of compartmentation of glycolysis in procyclic form Trypanosoma brucei: RNA interference studies of PEX14, hexokinase, and phosphofructokinase.

Trypanosoma brucei and related organisms contain an organelle evolutionarily related to peroxisomes that sequesters glycolysis, among other pathways. We have shown previously that disruption of protein import into this organelle, the glycosome, can be accomplished through RNA interference (RNAi)-mediated knockdown of the peroxin PEX14. Decreased PEX14 in turn leads to cell death, which, at least in the procyclic stage, can be triggered by the presence of glucose. Here we show that fructose, which is taken up and metabolized by procyclic form T. brucei, and glycerol, which interfaces with the glycosomal glycolytic pathway, are also toxic during PEX14 RNAi. Earlier computer modeling studies predicted that glycolysis would be toxic to T. brucei in the absence of glycosomal compartmentation because of the intrinsic lack of feedback regulation of the parasite hexokinase and phosphofructokinase. To further test this hypothesis, we performed double RNAi, targeting hexokinase and PEX14. Knockdown of hexokinase rescued PEX14 knockdown cells from glucose toxicity, even though glycosomal proteins continue to be mislocalized to the cytosol. Knockdown of phosphofructokinase was benign in the absence of glucose but toxic in the presence of glucose. When PEX14 and phosphofructokinase mRNAs were jointly targeted for RNAi, glycerol remained toxic to the parasites. Taken together, these data indicate that the glycosome provides significant, but not complete, protection of trypanosomes from the dangerous design of glycolysis.

Animals↗

PubMed2005

Paradigms for conditional expression of RNA interference molecules for use against viral targets.

The rapid increase in the study of small interfering RNA (siRNA) as a means to decrease expression of targeted genes has led to concerns about possible unexpected consequences of constitutive siRNA expression. We therefore devised a conditional siRNA expression system in which siRNA targeting hepatitis C virus (HCV) would be produced in response to HCV. We found that HCV acts via NFkappaB to stimulate the HIV long terminal repeat (LTR) as a promoter. We exploited this observation by designing conditional siRNA transcription constructs to be triggered by HCV-induced activation of NFkappaB. These were delivered by using highly efficient recombinant Tag-deleted SV40-derived vectors. Conditional activation of HIV-LTR and consequent siRNA synthesis in cells expressing HCV were observed. HCV-specific RNAi decreased HCV RNA greatly within 4 days, using transient transfection of the whole HCV genome as a model of acute HCV entry into transduced cells. We then tested the effectiveness of rSV40-delivered anti-HCV siRNA in cells stably transfected with the whole HCV genome to simulate hepatocytes chronically infected with HCV. There is considerable need for regulated production of siRNAs activated by a particular set of conditions (HCV in this case) but quiescent otherwise. Approaches described here may serve as a paradigm for such conditional siRNA expression.

Animals↗

PubMed2005

One-oligonucleotide method for constructing vectors for RNA interference.

AIM: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. METHODS: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP, were cloned into plasmids driven by RNA polymerase III promoter H1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. RESULTS: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. CONCLUSION: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.

Base Sequence↗

PubMed2005

[Inhibition of HBs-GFP fusion gene expression by RNA interference].

OBJECTIVE: To develop an effective report gene system to test the effect of small interfering RNA (siRNA). METHODS: HBV S gene was fused with enhanced green fluorescent protein (EGFP) gene to form HBs-GFP and the plasmid containing HBs-GFP was constructed. A vector expressing small hairpin RNA (shRNA) pAVU6 + 4sh357 was also constructed. Two plasmids were co-transfected into HepG2 cells transiently. The fluorescence of HBs-GFP was detected by fluorescence-activated cell sorting (FACS). The mRNA expression in HepG2 cells was detected by conventional RT-PCR and real-time PCR. RESULTS: siRNA inhibited the expression of HBs-GFP 72 hours post transfection. The fluorescence of HBs-GFP in HepG2 cells treated with pAVU6+4sh357 was reduced by 55.4% compared with that of controls. The HBs-GFP expression in HepG2 cells treated with pAVU6+4sh357 was reduced by 76.3% and 90% as measured with conventional RT-PCR and real-time PCR, respectively. CONCLUSION: This investigation demonstrated siRNA derived from shRNA expression vectors can inhibit the expression of HBs-GFP in HepG2 cells.

Carcinoma, Hepatocellular↗

PubMed2006

[Suppression of MDR1 expression and restoration of sensitivity to chemotherapy in multidrug-resistant hepatocellular carcinoma cell line Bel7402/5-Fu by RNA interference].

OBJECTIVE: To investigate the suppression of MDR1 and P-glycoprotein induced by small interfering RNA and the restoration of sensitivity to chemotherapeutic drugs in multidrug-resistant hepatocellular carcinoma cell line Bel7402/5-Fu. METHODS: MDR1j targeted small interfering RNA duplexes were introduced into multidrug-resistant hepatocellular carcinoma cell line Bel7402/5-Fu. The suppression of MDR1 and its gene product P-glycoprotein was examined by RT-PCR and Western blot. MTT assay was performed to measure the reverse effect of small interfering RNA based on the results of IC50. Cell apoptosis was assessed by flow cytometry after various cell lines were treated with chemotherapeutic drugs. RESULTS: The overexpression of MDR1 and P-glycoprotein was suppressed efficiently by the introduction of small interfering RNA, which caused sequence-specific gene silence. The level of MDR1 in the transfected Bel7402/5-Fu cells reduced to 22.55% and P-glycoprotein to 25.49% compared with those of the controls. The apoptosis rate of Bel7402/5-Fu cells increased significantly in the siRNA group during the chemotherapy (P<0.01). Their resistance to 5-Fu was reversed by 14.88 folds, which indicated the restoration of sensitivity to drugs. CONCLUSION: Small interfering RNA can inhibit MDR1 expression effective and reverse the multidrug resistance mediated by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Stable RNA interference of host thrombospondin-1 blocks Trypanosoma cruzi infection.

Interactions between Trypanosoma cruzi and the extracellular matrix play an important role in cellular invasion. Here we show that T. cruzi increases the levels of thrombospondin-1 (TSP-1) expression in host cells during early infection. Stable RNA interference of host cell TSP-1 knocks down the levels of TSP-1 transcripts and protein expression in mammalian cells causing inhibition of T. cruzi infection. Addition of TSP-1 to these cells restores infection. Thus, host TSP-1, regulated by the parasite, plays a crucial role in early infection. This is the first report showing that a human parasite modulates TSP-1 expression to facilitate infection.

Animals↗

PubMed2004

The role of RNA interference in heterochromatic silencing.

Soon after its discovery 75 years ago, heterochromatin, a dense chromosomal material, was found to silence genes. But its importance in regulating gene expression was controversial. Long thought to be inert, heterochromatin is now known to give rise to small RNAs, which, by means of RNA interference, direct the modification of proteins and DNA in heterochromatic repeats and transposable elements. Heterochromatin has thus emerged as a key factor in epigenetic regulation of gene expression, chromosome behaviour and evolution.

Animals↗

PubMed2024

Antiviral RNA interference inhibits virus vertical transmission in plants.

Known for over a century, seed transmission of plant viruses promotes trans-continental virus dissemination and provides the source of infection to trigger devastating disease epidemics in crops. However, it remains unknown whether there is a genetically defined immune pathway to suppress virus vertical transmission in plants. Here, we demonstrate potent immunosuppression of cucumber mosaic virus (CMV) seed transmission in its natural host Arabidopsis thaliana by antiviral RNA interference (RNAi) pathway. Immunofluorescence microscopy reveals predominant embryo infection at four stages of embryo development. We show that antiviral RNAi confers resistance to seed infection with different genetic requirements and drastically enhanced potency compared with the inhibition of systemic infection of whole plants. Moreover, we detect efficient seed transmission of a mutant CMV lacking its RNAi suppressor gene in mutant plants defective in antiviral RNAi, providing further support for the immunosuppression of seed transmission by antiviral RNAi.

Plant Diseases↗

PubMed2006

Stable RNA interference of ErbB-2 gene synergistic with epirubicin suppresses breast cancer growth in vitro and in vivo.

Overexpression of human epidermal growth factor receptor-2 (Her2, ErbB-2) contributes to the progression and metastasis of breast cancer, implying that Her2 gene is a suitable target of RNA interference (RNAi) for breast cancer therapy. Here, we employed plasmid-mediated expression of 2 different Her2-shRNAs (pU6-Her2shRNAs) efficiently silenced the target gene expression on Her2 expressing SKBR-3 breast cancer cells in both mRNA and protein levels. Consequently, pU6-Her2shRNA increased apoptosis and reduced proliferation of SKBR-3 cells assayed by TUNEL and MTT, respectively. In vivo, intra-tumor injection of pU6-Her2shRNA inhibited the growth of SKBR-3 tumors inoculated subcutaneously in nude mice. Furthermore, pU6-Her2shRNA synergized the tumor suppression effect of epirubicin to SKBR-3 cells in vitro and implanted subcutaneously in nude mice. Therefore, we concluded that stable silencing of Her2 gene expression with plasmid expressing shRNA may hold great promise as a novel therapy for Her2 expressing breast cancers alone or in combination with anthracycline chemotherapy.

Animals↗

PubMedDate not supplied

[Inhibition of the motility of melanoma cells using interference RNA against CD9].

INTRODUCTION: Although 65 % of the deaths caused by skin cancer are due to melanomas, the possible causes that may explain the aggressiveness of this type of tumor are still unknown. Different approaches have been used to try to find an effective treatment, but they have been unsuccessful. Interference RNA (iRNA) is an essential technique for this research. With this technique, we can selectively "knock down" or silence protein expression. The tetraspanins CD9 and CD151 are molecules involved in cell motility, including melanoma cells. MATERIAL AND METHODS: A375 melanoma cell line, anti-CD9 and anti-CD151 monoclonal antibodies, and iRNA against CD9 and CD151. Immunofluorescence techniques, flow cytometry, cell transfection, cell selection with magnetic beads and evaluation of cell migration in a wound-healing model. RESULTS: The A375 cells express CD9 and CD151. By using iRNA against CD9 and CD151, we managed to inhibit the expression of these proteins. The cells transfected with iRNA against CD9 showed significant inhibition of their motility. DISCUSSION: We managed to silence the expression of CD9 and CD151 using iRNA techniques in the A375 melanoma cell line. The reduction in CD9 caused the inhibition of cell motility, while the interference with CD151 expression had a more moderate effect. This data indicates that the knockdown of tetraspanins may be a future target for the treatment of melanoma.

Antigens, CD↗

PubMed2001

RNA interference of a trypanosome topoisomerase II causes progressive loss of mitochondrial DNA.

We studied the function of a Trypanosoma brucei topoisomerase II using RNA interference (RNAi). Expression of a topoisomerase II double-stranded RNA as a stem-loop caused specific degradation of mRNA followed by loss of protein. After 6 days of RNAi, the parasites' growth rate declined and the cells subsequently died. The most striking phenotype upon induction of RNAi was the loss of kinetoplast DNA (kDNA), the cell's catenated mitochondrial DNA network. The loss of kDNA was preceded by gradual shrinkage of the network and accumulation of gapped free minicircle replication intermediates. These facts, together with the localization of the enzyme in two antipodal sites flanking the kDNA, show that a function of this topoisomerase II is to attach free minicircles to the network periphery following their replication.

Animals↗

PubMed2005

The purine synthesis gene Prat2 is required for Drosophila metamorphosis, as revealed by inverted-repeat-mediated RNA interference.

PRAT (phosphoribosylamidotransferase; E.C. 2.4.2.14) catalyzes the first reaction in de novo purine nucleotide biosynthesis. In Drosophila melanogaster, the Prat and Prat2 genes are both highly conserved with PRAT sequences from prokaryotes and eukaryotes. However, Prat2 organization and expression during development is different from Prat. We used RNA interference (RNAi) to knock down expression of both Prat and Prat2 to investigate their functions. Using the GAL4-UAS system, Prat RNAi driven by Act5c-GAL4 or tubP-GAL4 causes variable pupal lethality (48-100%) and approximately 50% female sterility, depending on the transgenic strains and drivers used. This observation agrees with the phenotype previously observed for Prat EMS-induced mutations. Prat2 RNAi driven by Act5C-GAL4 or tubP-GAL4 also results in variable pupal lethality (61-93%) with the different transgenic strains, showing that Prat2 is essential for fly development. However, Prat2 RNAi-induced arrested pupae have a head eversion defect reminiscent of the "cryptocephal" phenotype, whereas Prat RNAi-induced arrested pupae die later as pharate adults. We conclude that Prat2 is required during the prepupal stage while Prat is more important for the pupal stage. In addition, Prat and Prat2 double RNAi results in more severe pupal lethal phenotypes, suggesting that Prat and Prat2 have partially additive functions during Drosophila metamorphosis.

Amidophosphoribosyltransferase↗

PubMed2005

Down-regulation of IL-8 expression in human airway epithelial cells through helper-dependent adenoviral-mediated RNA interference.

Interleukin (IL)-8 is a potent neutrophil chemotactic factor and a crucial mediator in neutrophil-dependent inflammation. Various cell types produce IL-8, either in response to external stimuli such as cytokines or bacterial infection, or after malignant transformation. Anti-IL-8 strategies have been considered for anti-inflammatory therapy. In this paper we demonstrate that the RNA interference technique can be used to efficiently down-regulate IL-8 protein expression in airway epithelial cells. We used a helper-dependent adenoviral vector to express a small hairpin (sh)RNA targeting human IL-8 in cultured airway epithelial cells (IB3-1, Cftr-/-; C38, Cftr-corrected) stimulated with TNF-alpha, IL-1beta or heat-inactivated Burkholderia cenocepacia. Stimulated IL-8 expression in IB3-1 and C38 cells was significantly reduced by shRNA expression. The shRNA targeting IL-8 had no effect on the activation of NF-kappaB, or on the protein levels of IkappaB or IL-6, suggesting that this anti-IL-8 strategy was highly specific, and therefore may offer potential for the treatment of inflammatory diseases.

Adenoviridae↗

PubMed2005

[Inhibition of vascular endothelial growth factor gene expression and proliferation of leukemia cells by RNA interference].

OBJECTIVE: To explore the feasibility of selective inhibiting VEGF expression using VEGF short hairpin RNA (shRNA) interference, and observe the effects of VEGF gene silencing on NB4 cells growth. METHODS: Three 19 bp reverse repeated motifs targeting exons 3, 4, 5 respectively of VEGF gene were synthesized and cloned into eukaryotic expression plasmid pGenesil-1 containing U6 shRNA promoter and termination signal of RNA polymerase. The recombinant plasmids pGenesil-VR1, pGenesil-VR2, pGenesil-VR3 and pGenesil-con (plasmid containing random DNA fragment) were transfected into NB4 cells respectively through lipofectamine reagent. The alteration of VEGF expression was examined by fluorescent real time RT-PCR and Western blot. The proliferation capacity of leukemia cells was measured by trypan blue exclusion, MTT assay, colony formation assay and cell cycles analysis. RESULTS: Recombinant plasmids containing three shRNAs and random fragment were successfully constructed and transfected into NB4 cells respectively by liposome-mediated gene transfer method. shRNA in pGenesil-VR3 cells knocked down the expression of VEGF mRNA and protein dramatically in a sequence-specific manner when compared with that of pGenesil-VR1, Genesil-VR2 and pGenesil-con. The NB4 cells transfected with pGenesil-VR3 (NB4-VR3) had a more significant decrease in proliferation ability than NB4 and that transfected with pGenesil-con (NB4-con). The colony forming efficiencies of NB4-VR3, NB4-con and NB4 cell were (13.3 +/- 3.8)%, (21.3 +/- 6.4)% and (24.5 +/- 5.2)%, respectively (P < 0.05). Higher G(1) and lower S proportion were found in cell cycle distribution in comparison with the control groups by FCM. CONCLUSIONS: The shRNA can efficiently suppress VEGF expression in NB4 cells. Selective VEGF gene silence can inhibit the malignant proliferation of leukemia cells.

Apoptosis↗